Structural and mechanistic aspects of flavoproteins: probes of hydrogen tunnelling

Abstract

At least half of all enzyme‐catalysed reactions are thought to involve a hydrogen transfer. In the last 10 years, it has become apparent that many of these reactions will occur, in part, or in full, by quantum mechanical tunnelling. We are particularly interested in the role of promoting vibrations on H transfer, and the Old Yellow Enzyme family of flavoproteins has proven to be an excellent model system with which to examine such reactions. In this minireview, we describe new and established experimental methods used to study H‐tunnelling in these enzymes and we consider some practical issues important to such studies. The application of these methods has provided strong evidence linking protein dynamics and H‐tunnelling in biological systems.

Monoamine Oxidases: The Biochemistry of the Proteins As Targets in Medicinal Chemistry and Drug Discovery

“…Kinetic isotope studies in the steady-state confirm that the hydrogen transfer step is ratelimiting for both enzymes [152,239,244,245,248], but the values are slightly smaller for MAO B where oxidation rates are not much faster than reduction. Detailed analysis of kinetic isotope effects and temperature dependence are consistent with hydrogen tunneling in MAO B [248,249]. When the kinetic isotope effects on k red for MAO A were determined, there was a clear decrease between pH 7.5 and 8 supporting with other experiments the conclusion that the substrate deprotonation with a pKa in that range is responsible for the pH dependence of the steady-state reaction [239].…”

Old Yellow Enzyme‐Catalyzed Dehydrogenation of Saturated Ketones

“…Thereby, most E. coli proteins were precipitated while the thermostable Gk OYE remained in the supernatant. As common among OYEs,11 the active site prosthetic flavin is non‐covalently bound, as shown by trichloroacetic acid precipitation. MS analysis of the supernatant revealed the active site flavin to be flavin mononucleotide (FMN, see Supporting Information), a finding subsequently corroborated by the elucidation of the X‐ray crystal structure of Gk OYE (see also Figure 3).…”

Sweating the assets of flavin cofactors: new insight of chemical versatility from knowledge of structure and mechanism

“…Unifying concepts have emerged, but controversies remain where the balance of available data do not provide unequivocal evidence in support of a generally accepted reaction mechanism. Notable have been the current and historical debates concerning mechanisms of amine oxidation [6], or C-H bond breakage [7], based on reasoning from structural, computational and kinetic/spectroscopic investigations. These are rehearsed in some detail elsewhere, and are therefore not the focus of this article.…”